This invention relates to an apparatus and process for effecting a plurality of sequential chemical syntheses in a plurality of reaction porous substrates.
Instrumentation for the automatic synthesis of peptides has been available since 1964, when Merrifield described the first automated solid phase peptide synthesizer. Since that time Merrifield-type peptide synthesizers employing Merrifield chemistry (Boc-protected amino acids, polystyrene based synthesis resins, deprotection with trifluoroacetic acid, cleavage of peptide from resin with hydrogen fluoride) have come into wide use. More recently, the so-called Fmoc method of synthesis, Atherton et al (1979) Bioorg. Chem 8, 351, (Fmoc-protected amino acids, protective acrylamide-silica gel based synthesis supports, deprotection with alkaline reagents, cleavage of peptides from supports with trifluoroacetic acid) have become popular. One characteristic that distinguishes the Merrifield and the Fmoc syntheses is that the former requires that reactions be carried out in a shaken reaction cell, whereas in the latter case the support can be packed into a column and reagents pumped through. The flow-through synthesis capabilities of the Fmoc method give it several advantages, which is one reason it has become increasingly popular in recent years.
Also in recent years there has been an increase in demand for small peptides of similar structure. One type of need is for epitope mapping of proteins, i.e., a search for the small regions (6-12 amino acids) of proteins that are antigenic sites for binding of antibodies; or immunogenic sites, which stimulate the immune response. Immunogenic peptides have the potential for use in making vaccines. One way to search for these sites in a protein containing, for example, 200 amino acids, is to synthesize a set of approximately 200 overlapping hexapeptides, each differing from its neighbor by a single amino acid. Other applications are the synthesis of analogs of a biologically active peptide, whether to find a more active peptide, or to determine which amino acids are responsible for activity, by systematic variation of the sequence. Synthesis of such large numbers of peptides one by one, such as those set forth below, even using a machine, is very time consuming.
______________________________________ ABCDEFGHIJKLMNOP ABCDEFG ABCDEFG ABCDE XBCDEFG AXCDEFG BCDEF AXCDEFG AYCDEFG CDEFG ABXDEFG AWCDEFG DEFG ABCXEFG AZCDEFG EFGHI ABCDXFG AQCDEFG ______________________________________
There have been several methods and devices described to speed up this process. One of the first is the "teabag" method of Houghton (1985), Proc. Natl. Sci. USA 82, pg. 531 where synthesis is carried out on resins in small porous bags, which are soaked in solutions of the appropriate activated amino acid. Several bags can be placed in a single reaction vessel, and by proper "mixing and matching", several similar peptides can be synthesized (on a 50-100 umole scale) simultaneously. This process has not been automated, however. Another method is the "polypropylene peg method" of Geysen et al, (1985) Proc. Natl. Acad. Sci. USA 82, 178-182 wherein very small quantities ( 0.1 umole) of peptide are synthesized on small polypropylene rods by dipping the rods into the appropriate solutions. The quantities that can be made are very small, and the process is not automated. Other multiple peptide synthesis systems are DuPont's RaMPS system, which is manual, and the method of Schnorrenberg et al (1989), Tetrahedon 45, pgs. 7759-7764. The latter device uses a robot arm to deliver reagents to synthesis support resins in wells of a microtitre plate and is purported to be capable of synthesizing 96 peptides at once.
The various available methods can be classified somewhat arbitrarily, an "macroscale" (&gt;10 umole), "microscale" ( 100 nmole) and "intermediate" (&lt;100 nmole to 10 umole). Virtually all of the macroscale methods, many of which are automated, involve synthesis of peptides on resins and other types of beads. The microscale methods usually involve synthesis of peptides on surfaces such as polyethylene rods. Because of the small amounts of peptides synthesized, usually no attempt is made to isolate the peptide and the subsequent assay reactions involving the peptides are done directly on the surface, such as by binding antibodies to the surface. Macroscale synthesizers are very efficient but generally permit synthesis of only one peptide at a time. The primary problem with microscale methods is that they permit synthesis of only very small amounts of peptides or of peptides of very poor quality, e.g., 50% to 80% yields per cycle as compared with 99% for many macroscale synthesizers.
Accordingly, it would be desirable to provide a method and apparatus for producing a multiplicity of peptides in the micro to intermediate scale range which is rapid and effects high yields of peptide product.